Coronary artery disease (CAD) is the number one cause of morbidity and mortality in the United States. Despite advances in medical therapies, some patients continue to develop refractory symptoms requiring more aggressive measures such as left ventricular assist device (LVAD) or orthotopic heart transplantation (OHT). Over the past decade, remarkable progress in recombinant DNA technology has enabled the development of molecular and cellular treatments for CAD. In particular, the field of cardiac gene therapy has evolved from in vitro studies to pre-clinical testing to multi-center trials. However, recent phase II/III trials have uncovered problems such as difficulties in controlling immunogenicity of adenoviral vectors, targeting expression to cardiac tissues, and in vivo monitoring of recombinant genes post delivery. Thus, the development of novel non-viral vectors that are safe and can yield targeted tissue delivery would be a major advance. Another significant advance will be the development of noninvasive techniques to assess gene expression, providing a new catalyst for the entire field of investigation. The aims of this proposal are to (1) develop non- immunogenic minicircle DNA vectors will significantly improve transfection efficiency in the heart, (2) understand the mechanisms of gene based therapy using integrated strategies of genetic and molecular assays, and (3) to evaluate the safety and efficacy in pre-clinical large animal models. At the end of 5 years, we hope to translate these findings to treatment of CAD patients with minicircle- based gene therapy in the future.